Sc 374429

Immunoprecipitation was performed according to the method described previously [39 (link)]. Lysates were pre-cleaned in lysis buffer with protein A/G agarose (Santa Cruz) for 1 h at 4°C and subsequently centrifuged. The supernatants were incubated with a precipitation antibody (either to CLDN4 (4D3), CLDN7 (SC-17670, Santa-Cruz), or GST (ab19256, Abcam)), and protein A/G agarose, for 3 h at 4°C. Precipitates were collected via centrifugation, washed five times with lysis buffer, solubilized with sample buffer (Sigma, 40 μg), and subjected to an immunoblot analysis with antibodies to CLDN4 (4D3), integrin β1 (SC-374429, Santa-Cruz), and/or CD44 (SC-7297, Santa-Cruz).

Total protein from tissue lysates of iPSCs or skin tissues was isolated using Radio-immunoprecipitation assay buffer (#89901; Thermo Fisher Scientific) containing 1% protease-inhibitor cocktail and 1% phenylmethylsulfonyl fluoride according to the manufacturer’s instructions. Proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA, USA). The membrane was blocked for 1 h in 10% skim milk and then incubated with primary antibodies against integrin β1 (1:100; #sc-374429; Santa Cruz Biotechnology, Dallas, TX, USA) and β-actin (1:10,000; #AB0035; Abways, Shanghai, China) overnight at 4 °C. The following day, the membrane was washed three times for 10 min each with PBS containing 0.1% Tween-20 and then incubated with Peroxidase-AffiniPure donkey anti-rabbit IgG (H + L) (1:2000; #711-035-152; Jackson ImmunoResearch) at 37 °C for 1 h, followed by three washes for 10 min each with PBS. The immunoreactive bands were visualized using a chemiluminescence substrate detection system (Tanon, Beijing, China).

MSC-exos, cells and kidney tissues were lysed in RIPA lysis buffer (ThermoFisher) and the protein concentration was detected using BCA assay (ThermoFisher). Protein samples were subjected to Bis-Tris Gel (Invitrogen) and transferred onto PVDF membranes (Millipore). Membranes were blocked in NcmBlot blocking buffer (NCM Biotech) for 10 min and incubated overnight with primary antibodies as follows: anti-CD9 (ab92726, Abcam), anti-CD63 (sc-5275, Santa Cruz), anti-Tsg101 (sc-7964, Santa Cruz) anti-Alix (sc-53540, Santa Cruz), anti-GM130 (12480, Cell Signaling Technology), anti-CD44 (ab189524, Abcam), anti-ICAM-1 (MA5407, Invitrogen), anti-VCAM-1 (39036, Cell Signaling Technology), anti-LFA-1 (ab13219, Abcam), anti-integrin α4 (8440, Cell Signaling Technology), anti-integrin β1 (sc-374429, Santa Cruz), anti-PCNA (sc-56, Santa Cruz), anti-CDK1 (sc-54, Santa Cruz), anti-caspase-3 (ab13847, Abcam), anti-Cyclin B1(4138, Cell Signaling Technology), anti-p53 (2524, Cell Signaling Technology), anti-Bcl-2 (sc-7382, Santa Cruz), and anti-Bax (sc-20067, Santa Cruz). Secondary antibodies were used for detection by an ECL advanced system (GE Healthcare). Intensity values expressed as the relative protein expression were normalized to β-actin (AB2001, Abways) or GAPDH (AB2000, Abways).